Human cytomegalovirus (HCMV) is a medically significant herpesvirus. Viral growth relies upon the proper production, localization, and function of its key proteins. The predominant products of the UL37 immediate early (IE) locus during HCMV infection are the essential UL37 exon 1 protein (pUL37x1) and a UL37 glycoprotein (gpUL37). Physical dissociation and differential trafficking are unique features of UL37 IE products, which appear to traffic to the ER and to mitochondria. This trafficking is sequential: gpUL37 is N-glycosylated and protease cleaved in the ER, so that its C-terminus is ER-retained while its N-terminus is imported into mitochondria where it inhibits apoptosis. Exceptionally few cellular mitochondrial proteins originate in the ER. The overall goal of this proposal is to examine the mechanistic basis for UL37 protein trafficking, processing, and functional role(s) during HCMV infection. We hypothesize that HCMV UL37 IE proteins traffic from the ER to mitochondria by targeting the mitochondria associated membrane (MAM) fraction and that the unique domains of UL37 isoforms result in differential trafficking and diverse functions. In Aim 1, we will test if UL37 protein trafficking through the secretory apparatus and mitochondria are interrelated. We will perform pulse-chase studies and subcellular localization of UL37 proteins in HCMV-infected cells as well as live cell imaging to determine if UL37 proteins traffic from ER to mitochondria. In Aim 2, we will examine post-translational processing of pUL37x1 and gpUL37, generate and assay UL37 mutants to determine which residues alter processing, trafficking, and function. In Aim 3, the function of HCMV UL37 proteins will be examined in the context of viral infection. We will complement, with the corresponding UL37 isoforms, an HCMV UL37 exon 3 mutant, which is growth delayed in G0-human fibroblasts. The induction of cellular gene expression in parental or in UL37 mutant HCMV-infected differentiated cells will be analyzed by microarray analysis. A thorough understanding of the regulation of how the essential UL37 proteins traffic to their ultimate locations and their function in infected cells is of fundamental importance in understanding the HCMV life cycle and its pathogenesis. Deciphering UL37 protein trafficking may provide valuable insight into cellular ER to mitochondrial trafficking pathways and, ultimately, be used against HCMV for therapeutic benefit.